Distinct functions of BMP4 during different stages of mouse ES cell neural commitment

Kejing Zhang 1 Lingyu Li 1 Chengyang Huang 1 Chengyong Shen 1 Fangzhi Tan 1 Caihong Xia 1 PingyuLiu 1 Janet Rossant 0 Naihe Jing 1 0 Program in Developmental and Stem Cell Biology, Hospital for Sick Children Research Institute, Department of Molecular Genetics, University of Toronto, 555 University Avenue , Toronto , ON M5G 1X8, Canada 1 Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences , 320 Yue Yang Road, Shanghai 200031, China T N E M P O L E V E D - SUMMARY Bone morphogenetic protein (BMP) signaling plays a crucial role in maintaining the pluripotency of mouse embryonic stem cells (ESCs) and has negative effects on ESC neural differentiation. However, it remains unclear when and how BMP signaling executes those different functions during neural commitment. Here, we show that a BMP4-sensitive window exists during ESC neural differentiation. Cells at this specific period correspond to the egg cylinder stage epiblast and can be maintained as ESC-derived epiblast stem cells (ESD-EpiSCs), which have the same characteristics as EpiSCs derived from mouse embryos. We propose that ESC neural differentiation occurs in two stages: first from ESCs to ESD-EpiSCs and then from ESD-EpiSCs to neural precursor cells (NPCs). We further show that BMP4 inhibits the conversion of ESCs into ESD-EpiSCs during the first stage, and suppresses ESDEpiSC neural commitment and promotes non-neural lineage differentiation during the second stage. Mechanistic studies show that BMP4 inhibits FGF/ERK activity at the first stage but not at the second stage; and IDs, as important downstream genes of BMP signaling, partially substitute for BMP4 functions at both stages. We conclude that BMP signaling has distinct functions during different stages of ESC neural commitment. INTRODUCTION During mouse embryonic development, the embryonic day (E) 3.5 blastocyst becomes a vesicular structure comprising an inner cell mass (ICM) inside the trophectoderm (Gardner and Beddington, 1988). By the time of implantation (E4.0-E4.5), the pluripotent ICM cells differentiate to form the primitive endoderm and epiblast (Gardner and Rossant, 1979). From E4.5 to E5.5, the blastocyst gives rise to a cup-shaped structure called the egg cylinder, and the epiblast at this stage is composed of a columnar epithelial monolayer of pluripotent cells (Coucouvanis and Martin, 1995; Snow, 1977). Gastrulation then commences with the formation of the primitive streak at approximately E6.5, through which epiblast cells ingress to form the mesoderm and the endoderm. The cells that remain in the anterior of the epiblast form the ectoderm (Lu et al., 2001; Tam and Loebel, 2007). Neural induction is the process during which part of the ectoderm is specified and becomes the embryonic neural plate. This process is generally thought to occur at the onset of gastrulation (Hemmati-Brivanlou and Melton, 1997b). To date, knowledge of the molecular mechanisms governing these events in the mouse embryo is limited owing to the small size, complexity and inaccessibility of the early postimplantation embryo, and to the rapid pace of cell proliferation. *These authors contributed equally to this work Author for correspondence () Mouse embryonic stem cells (ESCs) are immortal cell lines derived mainly from the ICM of peri-implantation blastocysts (Brook and Gardner, 1997; Evans and Kaufman, 1981; Martin, 1981). The lineage restriction of mouse ESCs is identical to that of the epiblast progenitors in the ICM, suggesting that ESCs might represent an in vitro model of early epiblast development (Rossant, 2008). However, recent evidence has shown that ESCs are not homogenous and appear to be in a metastable state, shifting between ICM- and epiblast-like states while remaining pluripotent (Chambers et al., 2007; Hayashi et al., 2008; Toyooka et al., 2008). Epiblast stem cells (EpiSCs) have been isolated from the epithelialized epiblast of mouse and rat egg cylinders (Brons et al., 2007; Tesar et al., 2007). EpiSCs can be maintained as stable cell lines in the presence of FGF and activin, and they are capable of differentiating into three germ layers in vitro and of forming teratomas. On the basis of their developmental potential, EpiSCs are believed to resemble pluripotent progenitors in the late epiblast layer of the post-implantation mouse embryo (Brons et al., 2007). However, it is unclear whether ESC neural differentiation in vitro recapitulates the different stages of the developmental process from the ICM to neuroectoderm in vivo (Rossant, 2008; Silva and Smith, 2008). Bone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF) superfamily (Shi and Massague, 2003). In the past decade, the default model has proposed that ectodermal cells give rise to neural tissue autonomously in the absence of inhibitory BMP signals, whereas BMP activity directs ectoderm to become epidermis (HemmatiBrivanlou and Melton, 1997a). Although this default model has been challenged by studies in Xenopus and chick embryos (De Robertis and Kuroda, 2004; Linker and Stern, 2004; Wilson and Edlund, 2001), recent studies have shown that the complete inhibition of BMP signaling is sufficient to induce neural tissue (Di-Gregorio et al., 2007; Khokha et al., 2005; Reversade and De Robertis, 2005). In mouse ESCs, BMP signaling is crucial for maintaining pluripotency (Kawasaki et al., 2000; Tropepe et al., 2001; Ying et al., 2003a), and also has negative effects on neural differentiation (Kawasaki et al., 2000; Tropepe et al., 2001; Ying et al., 2003b). It has been reported that BMP4-induced ID proteins can inhibit ESC entry into the neural lineage, and can sustain ESC self-renewal in collaboration with LIF/STAT3 (Ying et al., 2003a). However, it remains unclear when and how BMP signaling regulates these different functions during ESC neural commitment. Here, we show that there is a BMP4-sensitive window during mouse ESC neural commitment. Cells at this stage correspond to the epiblast of the egg cylinder, and can be maintained as ESCderived EpiSCs (ESD-EpiSCs). We further show that BMP4 inhibits ESC neural differentiation in two phases: first, it inhibits the derivation of ESD-EpiSCs from mouse ESCs; and second, it suppresses the neural commitment of ESD-EpiSCs and promotes their non-neural differentiation. MATERIALS AND METHODS ESC culture and neural differentiation Mouse ESC lines R1, R1/E (ATCC), E14Tg2a, SOX-GFP ES cells (46C), and TAU-GFP ES cells (TK23) (Ying et al., 2003b) were used in this study. ESCs were maintained on mitomycin C-treated mouse embryonic fibroblasts (MEFs; feeders) in standard medium. ESC neural differentiation was induced as described previously (Watanabe et al., 2005). Derivation of ESD-EpiSCs and cell culture For derivation of ESD-EpiSCs, ESC aggregates were dissociated into a single-cell suspension after 5 minutes of (...truncated)